WO2010132879A2 - Polymères cationiques dégradables multicomposants - Google Patents

Polymères cationiques dégradables multicomposants Download PDF

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WO2010132879A2
WO2010132879A2 PCT/US2010/035127 US2010035127W WO2010132879A2 WO 2010132879 A2 WO2010132879 A2 WO 2010132879A2 US 2010035127 W US2010035127 W US 2010035127W WO 2010132879 A2 WO2010132879 A2 WO 2010132879A2
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group
compound
cells
polymer
polymers
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PCT/US2010/035127
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English (en)
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WO2010132879A3 (fr
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Jordan J. Green
Joel C. Sunshine
Nupura S. Bhise
Ron B. Shmueli
Stephany Y. Tzeng
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The Johns Hopkins University
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Priority to US13/320,621 priority Critical patent/US8992991B2/en
Publication of WO2010132879A2 publication Critical patent/WO2010132879A2/fr
Publication of WO2010132879A3 publication Critical patent/WO2010132879A3/fr
Priority to US13/272,042 priority patent/US9717694B2/en
Priority to US14/644,397 priority patent/US9884118B2/en
Priority to US15/645,337 priority patent/US10786463B2/en
Priority to US15/821,368 priority patent/US20180177881A1/en
Priority to US16/895,596 priority patent/US20200297851A1/en
Priority to US17/149,583 priority patent/US20210220287A1/en
Priority to US17/150,796 priority patent/US20210252154A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/10—Esters
    • C08F222/1006—Esters of polyhydric alcohols or polyhydric phenols
    • C08F222/102—Esters of polyhydric alcohols or polyhydric phenols of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • PEI polyethylenimine
  • PEI also is particularly effective at promoting endosomal escape of PEI/DNA particles through the proton sponge mechanism.
  • This mechanism is critical in preventing lysosomal degradation of the DNA and to enable efficient delivery of the DNA to the cytoplasm.
  • This endosomal escape mechanism has been used in the design of other synthetic gene delivery polymers, including polylysine-based polymers that contain an imidazole group in the side chain. See D. Putnam, et al., Proc. Natl. Acad. Sa.
  • PEI shows promise compared to other biomaterials, it also leads to significant cytotoxicity, see S. M. Moghimi, et al., MoI. Ther. 11 :990-5 (2005), and has lower effectiveness than viral methods.
  • poly(beta-amino ester)s are poly(beta-amino ester)s, see J. J. Green, et al. , Ace. Chem. Res. 41 :749-759 (2007), which are useful due to their ability to bind DNA, promote cellular uptake, facilitate escape from the endosome, and allow for DNA release in the cytoplasm. See D. M. Lynn and R.
  • poly(beta-amino ester)s are readily biodegradable due to their ester linkages, which reduces cytotoxicity.
  • Another approach to increase gene delivery effectiveness while reducing cytotoxicity involves adding bioreducible linkages to polymers. Disulfide linkages have been added to PEI to produce bioreducible versions with lower cytotoxicity than high molecular weight versions of the parent polymer. M. A Gosselin, et al.,
  • IMR-90 human primary fibroblasts can be reprogrammed to induced pluripotent stem cells with integrating viruses. See
  • the presently disclosed subject matter includes a compound of formula (I): wherein: n is an integer from 1 to 10,000; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkyl thio ether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or
  • each R group is different
  • each R" group is different
  • each R" group is not the same as any of R', R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 ;
  • the R" groups degrade through a different mechanism than the ester- containing R groups, wherein the degradation of the R" group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
  • the compound of formula (I) comprises a substructure of a larger cross- linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I); and pharmaceutically acceptable salts thereof.
  • the presently disclosed compounds of formula (I) are useful for delivering a therapeutic agent to a cell, a specific cell line, a tissue, or an organism.
  • the therapeutic agent can include a gene, DNA, RNA, siRNA, miRNA, isRNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, and a small molecule drug.
  • the presently disclosed subject matter provides a method of treating a disease or condition, the method comprising administering to a subject in need of treatment thereof, a compound of formula (I) further comprising a therapeutic agent effective for treating the disease or condition.
  • Diseases that can be treated by the presently disclosed methods include, but are not limited to, a cancer, including brain cancer (including Glioblastoma Multiforme), lung cancer, breast cancer, prostate cancer, colorectal cancer, and other cancers; cardiovascular diseases; infectious diseases; ophthalmic diseases, including age-related macular degeneration.
  • the presently disclosed subject matter includes an in vitro kit comprising a compound of formula (I).
  • the presently disclosed subject matter includes a biomedical device, such as a stent or a stent-like device, comprising a compound of formula (I) or an article coated with one or more compounds of formula (I) alone or in combination with one or more commercially available and/or FDA-approved polyelectrolyte.
  • the presently disclosed subject matter provides a method of forming a tissue scaffolding structure, the method comprising implanting into a subject a polymeric matrix comprising a compound of formula (I).
  • the implant can include one or more cells selected from the group consisting of hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal- hypothalamic-pituitary axis, heart muscle cells, epithelial cells, kidney tubular cells, kidney basement cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, smooth and skeletal muscle cells, cells from the retina and other parts of the eye, stem cells, induced pluripotent stem cells, and three-dimensional organoids.
  • the presently disclosed subject matter provides a nanoparticle or microparticle comprising a compound of formula (I).
  • FIGS. la-Id are representative synthesis schemes of representative embodiments of the presently disclosed degradable gene delivery polymers:
  • (a) 1,4- butanediol diacrylate reacts with l-amino-5-pentanol to form an acrylate-terminated poly(beta-amino ester) precursor.
  • This precursor reacts with: (b) l-(3-aminopropyl)- 4-methylpiperazine to form Poly 1;
  • (d) 4-aminophenyl disulfide to form Poly 3;
  • FIG. 2 shows the gene delivery efficacy of Poly 1, Poly 2, Poly 3, compared to commercial reagents PEI and LIPOFECTAMINE 2000TM to COS-7 cells (above) and IMR-90 cells (below). Luciferase-encoding DNA is delivered and expression is measured as relative light units per gram protein. Polymer to DNA weight ratio (w/w) tunes gene delivery efficacy. Delivery is high even in the presence of serum proteins.
  • the ratios 1.05: 1 and 1.2:1 refer to polymerization conditions as described herein and wt/wt is the weight ratio of polymer to DNA.
  • Graphs show mean+SD, n ⁇ 4;
  • FIG. 3 demonstrates that genes encoding green fluorescent protein are delivered to primary cells in three-dimensional mammary epithelial organoids by the presently disclosed polymer designated B4-S5-E9 (also referred to herein as Poly 3);
  • FIG. 4 shows that the disulfide end-group, designated herein as E9, makes biphasic polymer designated B4-S5-E9 more effective than polymers designated B4- S5-E7 or B4-S5-E8, which have difference end groups designated E7 and E8 herein, at transfecting primary cells in three-dimensional organoids (shown in FIG. 3);
  • FIG. 5 shows gene delivery with a series of presently disclosed polymers comparable to FUGENE HD® in (top) Hl 46 and (bottom) H446 lung cancer cells;
  • FIG. 6 shows gene delivery of presently disclosed polymers designated as B4- S5-E10 to EPH4 mammary epithelial cells as compared to commercially available reagent FUGENE HD® (% GFP positive as determined by FACS);
  • FIG. 7 demonstrates the ability of a presently disclosed polymer designated B4-S5-E10 (60 w/w) for transfecting Glioblastoma Multiforme (GBM) cells as measured by flow cytometry and Green Fluorescent Protein (GFP) expression.
  • BBM Glioblastoma Multiforme
  • FIG. 8 shows a presently disclosed polymer (B4-S5-E10 (60 w/w)) and GBM cells by microscopy.
  • the Green Fluorescent Protein (GFP) image shows that many of these cells actively transcribe and translate this DNA to generate the GFP expression.
  • the delivered plasmid could encode various shRNA molecules that are active within the cell instead of or in addition to GFP. This is an indirect method of RNA delivery.
  • FIG. 9 shows analytical data characterizing the synthesis of generic base polymer, i.e., a precursor, designated B4-S5;
  • FIG. 10 shows six curves from a single gel permeation chromatography run.
  • Each bioreducible base polymer (5 mg/mL) was run with or without a reducing agent, dithiothreitol (DTT), at 5 mM concentration.
  • DTT dithiothreitol
  • FIG. 11 shows a competitive binding assay demonstrating that addition of glutathione to a bioreducible polymer significantly reduces the binding affinity of the polymer to DNA. This property helps the particles unpack more efficiently inside cells to release active agents and cargos.
  • the assay compares the quenching of Yo- Pro-1 :DNA complexes over various polymer concentrations;
  • FIG. 12 shows bioreducible, multi-component polymers that were used for gene delivery at 600 ng/well DNA.
  • the best BSS based polymers (designated BSS- S4-E4 and BSS-S4-E1) obtained signals that were 215X and 50X higher than untreated wells, respectively.
  • end-capping generic base polymer B4-S5 with two reducible end groups made two new effective polymers, which combine hydrolytic degradation with disulfide reduction of the end groups.
  • These polymers have comparable efficacy to FUGENE HD® and LIPOFECTAMINE 2000TM, and other highly effective degradable polymers.
  • the best bioreducible polymer formulations demonstrate significantly reduced transfection in the presence of 5mM glutathione (all controls unaffected);
  • FIG. 13 is the titration of representative presently disclosed polymers (e.g., B4-S5-E9, B4-S5-E10, BSS-S5-E7, and the like) showing that they can buffer the pH of the endosomal compartment (pH ⁇ 6) as is needed to protect drug delivery agents and facilitate endosomal rupture through the proton sponge effect.
  • This characteristic is representative of other presently disclosed polymers, which have a range of buffering capacities. Buffering the endosome (or lack of buffering) is important to facilitate endosomal escape (or endosome targeting) and also to protect the agent being delivered;
  • FIG. 14 shows polymer/siRNA particle size (nm) as a function of formulation conditions (weight ratio polymer to RNA) for a representative presently disclosed polymer designated B5-S3-E9 (1.2:1 ratio for B5-S3).
  • the formulations of the particles can be tuned to vary biophysical properties of the particle and their release;
  • FIG. 15 shows the particle size/biophysical characterization of polymer/siRNA nanoparticles formed by a presently disclosed polymer designated BL2-S5-E10, 1.2:1.
  • Mean particle size 20 w/w polymer/siRNA 100 nm.
  • the lower panels show RNA encapsulation by the presently disclosed polymers and the resulting particle size distribution. Size depends on polymer type and formulation conditions;
  • FIG. 16 shows direct delivery of siRNA to brain cancer cells using polymeric nanoparticles comprising a presently disclosed polymer designated B5-S3-E9. Brain cancer cells containing FITC-labeled siRNA molecules are shown as bright regions on this image
  • FIG. 17 shows the molecular weights of ten B4-S5-based polymers including B4-S5-E9 and B4-S5-E10. Molecular weight is in Dalton (Da). Ratios are molar monomer ratios (B4:S5) used during polymer synthesis.
  • FIG. 18 shows the particle sizing data of nanoparticles formed by self- assembly of B4-S5 -based polymers including B4-S5-E9 and B4-S5-E10 with enhanced green fluorescent protein (EGFP) DNA.
  • the particles were sized using two techniques: Dynamic Light Scattering (intensity-weighted mean) and Nanoparticle Tracking Analysis (NTA). NTA was used to determine both the direct number- weighted mean and the mode;
  • FIG. 19 shows particle sizing data of nanoparticles formed by polymer B6-S4- E8 and a representative hydro philic/negatively charged synthetic peptide (11-mer that includes 5 glutamic acid residues). Peak of blue curves shows particle sizes of -100 nm for the particles when formulated at a 5 : 1 polymer to peptide mass ratio. Relative particle concentration for alternative formulations are also shown (Dark blue is a 1 : 1 ratio, Green is a 10:1 ratio); FIG. 20 shows particle sizing data of nanoparticles formed by polymer BLl-
  • Peak of number distribution of nanoparticles is 74 nm for a polymer to peptide mass ratio of l :l;
  • FIG. 21 shows transfection of IMR90s 48 hours post addition of nanoparticles. GFP expression is shown on the left panels and cell viability on the right.
  • Polymer formulations are B4-S4-E7 1.2: 1 100 w/w, 90 °C (top panels) and B5-S3-E7 1.05:1 100 w/w 90 °C (bottom panels);
  • FIG. 22 shows transfection of a retinal neuron with a representative polymer (B4-S4-E8 (60 w/w)). GFP is expressed brightly and morphological structures are good;
  • FIG. 23 shows transfection of a luciferase gene across many representative polymers. Each polymer is able to form nanoparticles that deliver genes to COS -7 cells. The polymers were synthesized at 90 °C unless indicated as 40 °C. Tuning the polymer backbone monomer, side group monomer, terminal group monomer, monomer ratio during synthesis, synthesis temperature, and nanoparticle formulation ratio (w/w) each independently varies overall gene delivery efficacy;
  • FIG. 24 shows transfection of a luciferase gene across many representative polymers. Each polymer is able to form nanoparticles that deliver genes to primary human fibroblasts, IMR-90s. The polymers were synthesized at 90 °C unless indicated as 40 °C. Tuning the polymer backbone monomer, side group monomer, terminal group monomer, monomer ratio during synthesis, synthesis temperature, and nanoparticle formulation ratio (w/w) each independently varies overall gene delivery efficacy.
  • FIGS. 5, 6, 23, and 24, demonstrate that representative embodiments of the presently disclosed polymers also can exhibit cell-type specificity; and FIG. 25 shows polymer/peptide particle size depends on polymer structure, formulation conditions including buffer, and peptide that is being encapsulated.
  • C refers to peptide NGRKACLNP ASPIVKKIIEKMLNS and "P” refers to peptide LRRFSTMPFMFCNINNVCNF.
  • the presently disclosed subject matter generally provides multicomponent degradable cationic polymers.
  • the presently disclosed polymers have the property of biphasic degradation. Modifications to the polymer structure can result in a change in the release of therapeutic agents, which can occur over multiple time scales.
  • the presently disclosed polymers include a minority structure, e.g., an endcapping group, which differs from the majority structure comprising most of the polymer backbone.
  • the bioreducible oligomers form block copolymers with hydrolytically degradable oligomers.
  • the end group/minority structure comprises an amino acid or chain of amino acids, while the backbone degrades hydrolytically and/or is bioreducible.
  • small changes in the monomer ratio used during polymerization, in combination with modifications to the chemical structure of the end-capping groups used post-polymerization, can affect the efficacy of delivery of a therapeutic agent, including, but not limited to a gene, to a target.
  • changes in the chemical structure of the polymer either in the backbone of the polymer or end-capping groups, or both, can change the efficacy of gene delivery to a cell, e.g., a cancerous fibroblast line or a human primary fibroblast.
  • small changes to the molecular weight of the polymer or changes to the endcapping groups of the polymer, while leaving the main chain, i.e., backbone, of the polymer the same, can enhance or decrease the overall delivery of the gene to a cell.
  • the "R" groups that comprise the backbone or main chain of the polymer can be selected to degrade via different biodegradation mechanisms within the same polymer molecule. Such mechanisms include, but are not limited to, hydro lytic, bioreducible, enzymatic, and/or other modes of degradation.
  • compositions can be prepared according to Scheme 1 :
  • Scheme 1 Representative synthesis scheme for preparing the presently disclosed cationic polymers having biphasic biodegradation.
  • At least one of the following groups R, R', and R" contain reducible linkages and, for many of the presently disclosed materials, additional modes of degradation also are present. More generally, R' can be any group that facilitates solubility in water and/or hydrogen bonding, for example, OH, NH 2 and SH. Representative degradable linkages include, but are not limited to:
  • end group structures i.e., R" groups in Scheme 1, for the presently disclosed cationic polymers are distinct and separate from the backbone structures (R) structures, the side chain structures (R'), and end group structures of the intermediate precursor molecule for a given polymeric material.
  • the presently disclosed subject matter includes a compound of formula (I):
  • n is an integer from 1 to 10,000;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkyl thio ether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl,
  • each R group is different
  • each R" group is different; (c) each R" group is not the same as any of R', R 1 , R 2 , R3, R4, R5, Re, R 7 ,
  • the R" groups degrade through a different mechanism than the ester- containing R groups, wherein the degradation of the R" group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
  • the compound of formula (I) comprises a substructure of a larger cross- linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I).
  • the compound of formula (I) is subject to the further proviso that if at least one R group comprises an ester linkage, then the R" groups impart one or more of the following characteristics to the compound of formula (I): independent control of cell-specific uptake and/or intracellular delivery of a particle; independent control of endosomal buffering and endosomal escape; independent control of DNA release; triggered release of an active agent; modification of a particle surface charge; increased diffusion through a cytoplasm of a cell; increased active transport through a cytoplasm of a cell; increased nuclear import within a cell; increased transcription of an associated DNA within a cell; increased translation of an associated DNA within a cell; increased persistence of an associated therapeutic agent within a cell, wherein the therapeutic agent is selected from the group consisting of DNA, RNA, a peptide or a protein.
  • any poly(beta-amino ester) specifically disclosed or claimed in U.S. patent no. 6,998,115; U.S. patent no. 7,427,394; U.S. patent application publication no. US2005/0265961; and U.S. patent publication no. US2010/0036084, each of which is incorporated herein by reference in its entirety, is explicitly excluded from the presently disclosed compounds of formula (I).
  • the reducible or degradable linkage comprising R, R', and R" is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the above- mentioned provisos.
  • n is an integer from 1 to 1,000; in other embodiments, n is an integer from 1 to 100; in other embodiments, n is an integer from 1 to 30; in other embodiments, n is an integer from 5 to 20; in other embodiments, n is an integer from 10 to 15; and in other embodiments, n is an integer from 1 to 10.
  • R" can be an oligomer as described herein, e.g., one fully synthesized primary amine-terminated oligomer, and can be used as a reagent during the second reaction step of Scheme I. This process can be repeated iteratively to synthesize increasingly complex molecules.
  • R" can comprise a larger biomolecule including, but not limited to, poly(ethyleneglycol) (PEG), a targeting ligand, including, but not limited to, a sugar, a small molecule, an antibody, an antibody fragment, a peptide sequence, or other targeting moiety known to one skilled in the art; a labeling molecule including, but not limited to, a small molecule, a quantum dot, a nanoparticle, a fluorescent molecule, a luminescent molecule, a contrast agent, and the like; and a branched or unbranched, substituted or unsubstituted alkyl chain.
  • PEG poly(ethyleneglycol)
  • a targeting ligand including, but not limited to, a sugar, a small molecule, an antibody, an antibody fragment, a peptide sequence, or other targeting moiety known to one skilled in the art
  • a labeling molecule including, but not limited to, a small molecule, a quantum dot, a nanoparticle, a
  • the branched or unbranched, substituted or unsubstituted alkyl chain is about 2 to about 5 carbons long; in some embodiments, the alkyl chain is about 6 to about 8 carbons long; in some embodiments, the alkyl chain is about 9 to about 12 carbons long; in some embodiments, the alkyl chain is about 13 to aboutl8 carbons long; in some embodiments, the alkyl chain is about 19 to about 30 carbons long; in some embodiments, the alkyl chain is greater than about 30 carbons long.
  • both R" groups i.e., the end groups of the polymer, comprise alkyl chains.
  • only one R" group comprises an alkyl chain.
  • at least one alkyl chain is terminated with an amino (NH 2 ) group.
  • the at least one alkyl chain is terminated with a hydroxyl (OH) group.
  • the PEG has a molecular weight of about 5 kDa or less; in some embodiments, the PEG has a molecular weight of about 5 kDa to about 10 kDa; in some embodiments, the PEG has a molecular weight of about 10 kDa to about
  • both R" groups comprise PEG.
  • only one R" group comprises PEG.
  • one R" group is PEG and the other R" group is a targeting ligand and/or labeling molecule as defined herein above.
  • one R" group is an alkyl chain and the other R" group is a targeting ligand and/or labeling molecule.
  • Representative monomers used to synthesize the presently disclosed cationic polymers include, but are not limited to, those provided immediately herein below.
  • end group structures (R") and the backbone structures (R) are defined as above and the side chain must contain at least one hydroxyl (OH) group.
  • the presently disclosed cationic polymer comprises a specific poly(ester amine) structure with secondary non-hydrolytic modes of degradation.
  • the cationic polymer comprises a polyester that degrades through ester linkages (hydrolytic degradation) that is further modified to comprise bioreducible groups as end (R”) groups.
  • bioreducible end groups in such embodiments include, but are not limited to:
  • the presently disclosed cationic polymer comprises a specific poly(ester amine alcohol) structure with secondary non-hydrolytic modes of degradation.
  • the cationic polymer comprises a specific structure where a polyester that degrades through ester linkages (hydrolytic degradation) is modified to contain bioreducible groups as end groups.
  • the presently disclosed cationic polymer comprises a specific poly(amido amine) structure having disulfide linking groups in the polymer backbone and an independent, non-reducible amine contacting group at the terminal ends of the polymer.
  • R 1 and R 2 are alkyl chains.
  • the alkyl chain is 1-2 carbons long; in some embodiments, the alkyl chain is 3-5 carbons long; in some embodiments, the alkyl chain is 6-8 carbons long; in some embodiments, the alkyl chain is 9-12 carbons long; in some embodiments, the alkyl chain is 13-18 carbons long; in some embodiments, the alkyl chain is 19-30 carbons long; and in some embodiments, the alkyl chain is greater than 30 carbons long
  • Suitable non-reducible amino R" groups for such embodiments include, but are not limited to:
  • the presently disclosed cationic polymers comprise a specific poly(amido amine alcohol) structure having disulfide linking groups in the polymer backbone and an independent non-reducible amine contacting group at the terminal ends of the polymer.
  • the presently disclosed cationic polymer comprises a copolymer of representative oligomers as described hereinabove.
  • Such embodiments include, but are not limited to, a poly(amido amine) structure having disulfides in the polymer backbone and an independently degradable (non-reducible) group at at least one end of the polymer.
  • Such embodiments also include using a cross-linker to add bioreducible linkages to hydrolytically degradable materials and also provide for higher molecular weight materials.
  • a representative example of this embodiment, along with suitable monomers is as follows:
  • the presently disclosed polymer is selected from the group consisting of:
  • R substituent groups that make up the presently disclosed polymers degrade via different biodegradation mechanisms within the same polymer. These biodegradation mechanisms can include hydrolytic, bioreducible, enzymatic, and/or other modes of degradation; (b) the ends of the polymer include a minority structure that differs from the majority structure that comprises most of the polymer backbone; (c) in several embodiments, the side chain molecules contain hydroxyl (OH)/alcohol groups.
  • the backbone is bioreducible and the end groups of the polymer degrade hydrolytically; (b) the backbone degrades hydrolytically and the end groups are bioreducible; and (c) hydrolytically degradable oligomers are cross- linked with a bioreducible cross-linker; (d) bioreducible oligomers form block copolymers with hydrolytically degradable oligomers; and (e) the end group/minority structure comprises an amino acid or chain of amino acids, whereas the backbone degrades hydrolytically and/or is bioreducible.
  • One way to synthesize the presently disclosed materials is by the conjugate addition of amine-containing molecules to acrylates or acrylamides.
  • This reaction can be done neat or in a solvent, such as DMSO or THF. Reactions can take place at a temperature ranging from about room temperature up to about 90 °C and can have a duration from about a few hours to about a few weeks.
  • a solvent such as DMSO or THF.
  • Reactions can take place at a temperature ranging from about room temperature up to about 90 °C and can have a duration from about a few hours to about a few weeks.
  • the presently disclosed methods can be used to create linear or branched polymers.
  • the molecular weight (MW) has a range from about 1 kDa to about 5 kDa, in other embodiments, the MW has a range from about 5 kDa to about 10 kDa, in other embodiments the MW has a range from about 10 kDa to about 15 kDa, in other embodiments, the MW has a range from about 15 kDa to about 25 kDa, in other embodiments, the MW has a range from about 25 kDa to about 50 kDa, and in other embodiments, the MW has a range from about 50 kDa to about 100 kDa.
  • the polymer forms a network, gel, and/or scaffold of apparent molecular weight greater than 100 kDa.
  • the presently disclosed subject matter provides the synthesis and characterization of a library of materials that are potentially useful for varied aspects of biomedical engineering.
  • the presently disclosed polymers can be applied in any field where polymers have been found useful including, but not limited to, drug delivery and nucleic acid delivery. Accordingly, in some embodiments, the presently disclosed polymers provide for efficient intracellular delivery of therapeutic agents, such as nucleic acids, proteins, and the like, into cells. Thus, the presently disclosed polymers are well suited for the efficient delivery of DNA for non- viral gene delivery applications.
  • the presently disclosed materials are useful for drug and gene delivery due, in part, to one or more of the following aspects: (a) an ability to bind and encapsulate cargos including, but not limited to, DNA, siRNA, peptides, and proteins; (b) an ability to facilitate uptake of the cargos into a range of cell types, with differential cell-type specificity.
  • the presently disclosed biodegradable, cationic polymers can be used to deliver one or more therapeutic agents, biomolecules or small molecules to a cell, tissue, and/or organism either in vitro or in vivo.
  • Representative therapeutic agents, biomolecules or small molecules include, but are not limited to, DNA, RNA (siRNA, miRNA, isRNA, agRNA, smRNA, and the like), nucleic acids, peptides, proteins, hydrophobic drugs, and small molecules.
  • Such embodiments can be used to treat various conditions or diseases including, but not limited to, cancer, including brain cancer (including Glioblastoma Multiforme), lung cancer, and other cancers; cardiovascular diseases; infectious diseases; ophthalmic diseases, including age-related macular degeneration.
  • the presently disclosed polymers also can be used as a genetic vaccine or as artificial antigen presenting cells; as an adjuvant; as an immunosuppressant; as an immune system modulator; as agents for cell targeting; for enhancement of crops; enhancement of animals; and other therapeutic use in humans.
  • the presently disclosed polymers are put together as a kit for the delivery of an agent, a nucleic acid, DNA, or RNA to a specific cell line or to any non-specified type of cell.
  • the presently disclosed polymers can be put together as a kit for the delivery of agents to specific cells to generate induced pluripotent stem cells. In some embodiments, the presently disclosed polymers can be put together as a kit for the delivery of agents to stem cells to control their growth, differentiation, and/or development.
  • biomaterials linear or branched oligomers, polymers, or cross-linked polymers
  • coatings for particles or devices via electrostatic or covalent interactions with the particles or surfaces.
  • Such devices include, but are not limited to, nanoparticles, microparticles, stents, stent-like devices, and the like.
  • Such coated devices also could be included in kits for screening or assay development.
  • the presently disclosed polymers can be used to coat surfaces for biomedical applications or environmental applications, including, but not limited to, coating devices such as stents, stent-like devices, implants, or other biomedical or drug delivery devices.
  • multilayered coatings comprising 1-10 polymer layers; in some embodiments, 11-20 polymer layers; in some embodiments, 21-30 polymer layers; in some embodiments, 31-50 polymer layers; in some embodiments, 51-100 polymer layers; and in some embodiments, greater than 100 polymer layers.
  • the presently disclosed polymers can be used as cosmetic products. In other embodiments, the presently disclosed polymers can be used as dental products
  • the degradation products or the presently disclosed polymers are bioactive.
  • the degradation products are drugs and/or pro-drugs.
  • the degradation products facilitate organelle targeting.
  • the degradation products facilitate nuclear targeting.
  • nanoparticles formed through the presently disclosed procedures that encapsulate active agents are themselves encapsulated into a larger microparticle or device.
  • this larger structure is degradable and in other embodiments it is not degradable and instead serves as a reservoir that can be refilled with the nanoparticles.
  • they can be constructed with multi-component degradable cationic polymers as described herein.
  • they can be constructed by FDA approved biomaterials, including, but not limited to, poly(lactic-co-glycolic acid) (PLGA).
  • PLGA poly(lactic-co-glycolic acid)
  • the nanoparticles are part of the aqueous phase in the primary emulsion.
  • the nanoparticles will remain in the aqueous phase and in the pores/pockets of the PLGA microparticles. As the microparticles degrade, the nanoparticles will be released, thereby allowing sustained release of the nanoparticles.
  • the nanoparticle targeting (through biomaterial selection, nanoparticle biophysical properties, and/or a targeting ligand) will be combined with transcriptional targeting.
  • Transcriptional targeting includes designing a promoter so that the delivered nanoparticles carrying a nucleic acid cargo are only active in the cells or tissue types of interest.
  • combinations of different genetic cargos and/or particles are co- delivered simultaneously to deliver nucleic acids that both: (1) induce apoptosis (genes for tumor necrosis factor-related apoptosis-inducing ligand(TRAIL), p53, and the like) and (2) cause differentiation of cancer stem cells (Bone morphogenetic protein 4 (BMP-4) DNA, Glycogen synthase kinase 3beta shRNA/siRNA, and the like).
  • BMP-4 cancer stem cells
  • These nucleic acids are driven by brain cancer specific promoters, such as Nestin and Sox-2 for brain cancer stem cells and Glial fibrillary acid protein (GFAP) for glia.
  • the presently disclosed subject matter also includes a method of using and storing the polymers and particles described herein whereby a cryoprotectant (including, but not limited to, a sugar) is added to the polymer and/or particle solution and it is lyophilized and stored as a powder.
  • a cryoprotectant including, but not limited to, a sugar
  • Such a powder is designed to remain stable and be reconstituted easily with aqueous buffer as one skilled in the art could utilize.
  • substituent refers to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained.
  • substituents may be either the same or different at every position.
  • the substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).
  • R groups such as groups R 1 , R 2 , and the like, or variables, such as "m” and "n"
  • R 1 and R 2 can be substituted alkyls, or R 1 can be hydrogen and R 2 can be a substituted alkyl, and the like.
  • R or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein.
  • certain representative “R” groups as set forth above are defined below.
  • hydrocarbon refers to any chemical group comprising hydrogen and carbon.
  • the hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions.
  • the hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.
  • Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
  • alkyl refers to C 1-20 inclusive, linear (i.e., “straight- chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n- undecyl, dodecyl, and the like, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • Lower alkyl refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C 1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Higher alkyl refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • alkyl refers, in particular, to C 1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C 1-8 branched-chain alkyls.
  • Alkyl groups can optionally be substituted (a "substituted alkyl") with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.
  • alkyl chain There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • Cyclic and “cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl.
  • Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • cycloalkylalkyl refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above.
  • alkyl group also as defined above.
  • examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentyl ethyl.
  • cycloheteroalkyl or “heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10- member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of N, O, and S, and optionally can include one or more double bonds.
  • the cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings.
  • Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the term heterocylic refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7- membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (
  • Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.
  • alkenyl refers to a monovalent group derived from a C 1-20 inclusive straight or branched hydrocarbon moiety having at least one carbon- carbon double bond by the removal of a single hydrogen atom.
  • Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, l-methyl-2-buten-1-yl, and the like.
  • cycloalkenyl refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond.
  • examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
  • alkynyl refers to a monovalent group derived from a straight or branched C 1-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of “alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propyne, 3-hexyne, and the like.
  • Alkylene refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • the alkylene group can be straight, branched or cyclic.
  • the alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents. " There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described.
  • An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.
  • aryl is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety.
  • the common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine.
  • aryl specifically encompasses heterocyclic aromatic compounds.
  • the aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others.
  • aryl means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.
  • the aryl group can be optionally substituted (a "substituted aryl") with one or more aryl group substituents, which can be the same or different, wherein "aryl group substituent" includes alkyl, substituted alkyl, alkenyl, alkynyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, haloalkyl, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, amino, alkylamino, dialkylamino, trialkylamino, acylamino, aroylamino, carbamoyl, cyano, alkylcarbamoyl, dialkylcarbamoyl, carboxyaldehyde, carboxyl, alkoxycarbonyl, carboxamide, aryl
  • substituted aryl includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.
  • heteroaryl and “aromatic heterocycle” and “aromatic heterocyclic” are used interchangeably herein and refer to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
  • Aromatic heterocyclic groups can be unsubstituted or substituted with substituents selected from the group consisting of branched and unbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide.
  • heterocyclic and aromatic heterocyclic groups that may be included in the compounds of the invention include: 3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine, 4-(bis-(4-fluorophenyl)methyl)piperazine, 4- (diphenylmethyl)piperazine, 4(ethoxycarbonyl)piperazine, 4- (ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine, 4-(l - phenyl ethyl)piperazine, 4-(l , 1 -dimethyl ethoxycarbonyl)piperazine, 4-(2-(bis-(2- propenyl) amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine, 4-(2- chlorophenyl)piperazine, 4(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine
  • a ring structure for example, but not limited to a 3 -carbon, a 4-carbon, a 5 -carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure.
  • n is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution.
  • Each R group if more than one, is substituted on an available carbon of the ring structure rather than on another R group.
  • the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:
  • a dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.
  • R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein).
  • acyl specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.
  • alkoxyl or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O-) or unsaturated (i.e., alkenyl-O- and alkynyl-O-) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C 1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo -hydro carbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxy, n-hexoxy, and the like.
  • alkoxyalkyl refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.
  • Aryloxyl refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl.
  • aryloxyl as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.
  • Alkyl refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl.
  • exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
  • Aralkyloxyl refers to an aralkyl-O- group wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxyl group is benzyloxyl.
  • Alkoxycarbonyl refers to an alkyl-O-CO- group.
  • exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-O-CO- group.
  • exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-O-CO- group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an amide group of the formula -CONH 2 .
  • Alkylcarbamoyl refers to a R'RN-CO- group wherein one of R and R' is hydrogen and the other of R and R' is alkyl and/or substituted alkyl as previously described.
  • Dialkylcarbamoyl refers to a R'RN-CO- group wherein each of R and R' is independently alkyl and/or substituted alkyl as previously described.
  • carbonyldioxyl refers to a carbonate group of the formula -O— CO— OR.
  • acyloxyl refers to an acyl-O- group wherein acyl is as previously described.
  • amino refers to the -NH 2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals.
  • amino refers to the -NH 2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals.
  • acylamino and alkylamino refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
  • alkylamino, dialkylamino, and trialkylamino refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom.
  • alkylamino refers to a group having the structure -NHR' wherein R' is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure - NR'R", wherein R' and R" are each independently selected from the group consisting of alkyl groups.
  • trialkylamino refers to a group having the structure -NR'R"R'", wherein R', R", and R'" are each independently selected from the group consisting of alkyl groups. Additionally, R', R", and/or R'" taken together may optionally be -(CH 2 ) k - where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamine
  • alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S-) or unsaturated (i.e., alkenyl-S- and alkynyl-S-) group attached to the parent molecular moiety through a sulfur atom.
  • thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • “Acylamino” refers to an acyl-NH- group wherein acyl is as previously described.
  • Aroylamino refers to an aroyl-NH- group wherein aroyl is as previously described.
  • carboxyl refers to the -COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.
  • halo refers to fluoro, chloro, bromo, and iodo groups.
  • hydroxyl refers to the -OH group.
  • hydroxyalkyl refers to an alkyl group substituted with an -OH group.
  • mercapto refers to the -SH group.
  • oxo refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.
  • nitro refers to the -NO 2 group.
  • thio refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
  • thiohydroxyl or thiol refers to a group of the formula -SH.
  • ureido refers to a urea group of the formula -NH — CO — NH 2 .
  • a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.
  • the term "monomer” refers to a molecule that can undergo polymerization, thereby contributing constitutional units to the essential structure of a macromolecule or polymer.
  • a "polymer” is a molecule of high relative molecule mass, the structure of which essentially comprises the multiple repetition of unit derived from molecules of low relative molecular mass, i.e., a monomer.
  • an "oligomer” includes a few monomer units, for example, in contrast to a polymer that potentially can comprise an unlimited number of monomers. Dimers, trimers, and tetramers are non-limiting examples of oligomers.
  • the term “nanoparticle,” refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and all integers and fractional integers in between).
  • the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm. In some embodiments, the nanoparticle has a diameter of about 200 nm. In other embodiments, the nanoparticle has a diameter of about 500 nm. In yet other embodiments, the nanoparticle has a diameter of about 1000 nm (1 ⁇ m). In such embodiments, the particle also can be referred to as a "microparticle.
  • the term "microparticle” includes particles having at least one dimension in the range of about one micrometer ( ⁇ m), i.e., 1 x 10 -6 meters, to about 1000 ⁇ m.
  • the term "particle” as used herein is meant to include nanoparticles and microparticles.
  • nanoparticles suitable for use with the presently disclosed methods can exist in a variety of shapes, including, but not limited to, spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism- shaped nanoparticles, and a plurality of other geometric and non-geometric shapes.
  • the presently disclosed nanoparticles have a spherical shape.
  • a subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term "subject.”
  • a "subject" can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes.
  • Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bo vines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like.
  • mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bo vines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; cap
  • an animal may be a transgenic animal.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a "subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease.
  • the terms “subject” and “patient” are used interchangeably herein.
  • association When two entities are “associated with” one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Preferably, the association is covalent. Desirable non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.
  • Biocompatible The term “biocompatible”, as used herein is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce inflammation or other such adverse effects.
  • Biodegradable As used herein, “biodegradable” compounds are those that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components preferably do not induce inflammation or other adverse effects in vivo. In certain preferred embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.
  • the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response.
  • the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like.
  • Peptide or “protein” A “peptide” or “protein” comprises a string of at least three amino acids linked together by peptide bonds.
  • the terms “protein” and “peptide” may be used interchangeably.
  • Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.
  • Polynucleotide or oligonucleotide Polynucleotide or oligonucleotide refers to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynyl cytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases,
  • Small molecule refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds.
  • Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin.
  • Known synthetic small molecules include, but are not limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.
  • the term "about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • the term "about” when used in connection with one or more numbers or numerical ranges should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth.
  • FIG. 1 Acrylate-terminated poly( 1,4-butanediol diacrylate-co-5-amino-l-pentanol) was first synthesized at two different acrylate monomer to amine monomer molar ratios, 1.05:1 and 1.2:1. For the 1.05:1 ratio, 3,532 mg of 1 ,4-butanediol diacrylate (17.8 mmol) was added to 1,754 mg of 5-amino-l-pentanol (17.0 mmol) and for the 1.2:1 ratio, 3,532 mg of 1,4-butanediol diacrylate (17.8 mmol) with 1,533 mg of 5- amino-1-pentanol (14.8 mmol). Reactions took place in DMSO (500 mg/mL) in glass vials in the dark under magnetic stirring for 48 hrs at 40°C.
  • DMSO 500 mg/mL
  • Polymers were analyzed by gel permeation chromatography using a Waters Breeze System and 3 Styragel Columns (7.8 x 300 mm) in series: HR 1, HR 3, and HR 4.
  • the eluent was 95% THF/5% DMSO/0.1 M piperidine and ran at 1 mL/min.
  • COS-7 and IMR-90 cells were grown following ATCC recommended protocols and reagents.
  • C0S-7s were grown in Dulbecco's Modified Eagle's Medium (DMEM, ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 units/mL of penicillin and streptomycin (Invitrogen).
  • IMR- 90s were grown in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 units/mL of penicillin and streptomycin. Cells were subcultured upon confluence and IMR-90s were used prior to passage eight.
  • Polyethylenimine/DNA particles were formed in a similar manner to the other polymers, except that they were formed at a w/w ratio of 1 (N/P-8) in 150-mM NaCl solution as has been previously described. See O. Boussif, et al., Proc. Natl. Acad. Sci. USA 92:7297-301 (1995); M. M. O. Sullivan, et al., Gene Ther. 10:1882-1890 (2003).
  • LIPOFECTAMINE 2000TM was used following the manufacturer instructions. Forty-eight hours post transfection, gene expression was measured using Bright-GIo luminescence assay kits (Promega), a Synergy 2 multilabel plate reader (Biotek), and a one second read time per well. Protein content per well was measured using the BCA protein assay kit (Pierce) and the Synergy 2 plate reader to measure absorbance at 562 nm.
  • Base acrylate-terminated polymers were synthesized via the conjugate addition of 5-amino-l-pentanol to an excess of 1 ,4-butanediol diacrylate in a manner similar to that previously described, but at a lower temperature and for a longer reaction time while being dissolved in DMSO. See J. J. Green, et al., Adv. Mater. 19:2836-2842 (2007); G. T. Switzerlandates, et al., MoI. Ther. 15: 1306-1312 (2007).
  • Polymerizations were performed at molar ratios of 1.05:1 and 1.2:1 at 40°C for 48 hrs. Subsequently, the polymers were end-modified by conjugate addition of l-(3-aminopropyl)-4-methylpiperazine (Poly 1), l-(3-aminopropyl)pyrrolidine (Poly 2), or 4-aminophenyl disulfide (Poly 3) to the base polymers at room temperature for 24 hrs (FIG. 1). Polymers were analyzed by gel permeation chromatography as shown in Table 1. For the 1.2: 1 molar ratio, polymers had a Mw of approximately 6 kDa.
  • polymeric particles formed with polymers synthesized at a ratio of 1.05:1 were generally more efficient for gene delivery than the same polymers formed at 1.2:1. This result is likely due to the higher MW of these polymers. In some cases, these changes were dramatic. For example, for Poly 1 at 20 w/w, the 1.05:1 ratio is more than 10-fold as effective as the 1.2:1 ratio with COS-7 cells and 400-fold more effective for IMR-90s (FIG. 2).
  • Certain end-modifying groups also appeared to show cell-type specificity.
  • Poly 3 at 60 w/w or 100 w/w has very high gene delivery to the COS-7 cancerous fibroblasts, but very poor delivery to the IMR-90 human primary fibroblasts.
  • Poly 3 (1.2: 1 ratio and 60 w/w) exhibits twice the gene expression in COS-7 cells, but over 200-fold less expression in IMR-90s.
  • Poly 1 or Poly 2 at a polymerization ratio of 1.05 : 1 and polymer to DNA weight ratio of 100 were the most effective.
  • LIPOFECTAMINE 2000TM is a leading commercially available lipid-based transfection reagent and the presently disclosed polymers can achieve comparable or higher delivery to both cancerous and primary cell types. Compared to 25 kDa branched polyethylenimine, a leading off-the-shelf gene delivery polymer, the presently disclosed polymers are up to 2 to 3 orders of magnitude more effective. In all cases, cells remained viable and comparable to untreated controls as determined by visual inspection and relative levels of protein content per well through the BCA assay.
  • Monomers BSS, S3, S4, S5, El, E2, E3, E4, E5, E6, E7, E8, E9, ElO, El l, E 12
  • controls LIPOFECTAMINE 2000TM and FUGENE HD®
  • Polymers were synthesized using a two-step procedure.
  • Acrylamide-terminated base polymer N,N'-bis(acrylyl)cystamine-co-4-amino-1- butanol etc.
  • monomer 1 1 molar ratios in a 9: 1 methanol: water mixture. Reactions took place in glass vials in the dark under nitrogen atmosphere with magnetic stirring for 5 days at 60°C.
  • polymers were synthesized using a two-step procedure. Polymers such as acrylate-terminated poly(1,4-butanediol diacrylate-co-5-amino-l-pentanol) were first synthesized at up to 7 different acrylate monomer to amine monomer molar ratios from 1.4: 1 to 1 : 1.4 (FIG. 9). Reactions took place in DMSO (500 mg/mL) in glass vials in the dark under magnetic stirring for 48 hrs at 40°C or neat at 90°C for 24 hours. Polymers were analyzed by gel permeation chromatography using a Waters Breeze System and 3 Styragel Columns (7.8 x 300 mm) in series: HR 1, HR 3, and HR 4. The eluent was 95% THF/5% DMSO/0.1 M piperidine and ran at 1 mL/min. B. Cell Culture
  • COS -7 cells (ATCC, Manassas, VA) were grown following ATCC recommended protocols and reagents. C0S-7s were grown in Dulbecco's Modified Eagle's Medium (DMEM, ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 units/mL of penicillin and streptomycin (Invitrogen).
  • DMEM Dulbecco's Modified Eagle's Medium
  • ATCC Dulbecco's Modified Eagle's Medium
  • ATCC fetal bovine serum
  • Invitrogen Invitrogen
  • Polymers at 100 mg/mL in DMSO were diluted in 25 mM sodium acetate buffer to concentrations that generate the varying polymer to DNA weight ratios (e.g., 20, 40, 60, and 100).
  • concentrations that generate the varying polymer to DNA weight ratios e.g., 20, 40, 60, and 100.
  • One hundred microliters of diluted polymer solution was mixed vigorously with 100 ⁇ L of DNA solution in a 96-well plate using a multichannel pipette. After 10 minutes wait time, 20 ⁇ L of each formulation was added to the cells that contained 100 ⁇ L of complete media per well. Particles were incubated with the cells for four hours and then removed with a 12-channel aspirator wand. Warm, complete media was added to the cells (100 ⁇ L/well) and they were allowed to grow for two days at 37°C and 5% CO 2 .
  • LIPOFECTAMINE 2000TM (Invitrogen) and FUGENE HD® (Roche) were used following the manufacturer instructions. Forty-eight hours post transfection, gene expression was measured using Bright-GIo luminescence assay kits (Promega), a Synergy 2 multilabel plate reader (Biotek), and a one second read time per well. Protein content per well was measured using the BCA protein assay kit (Pierce) and the Synergy 2 plate reader to measure absorbance at 562 nm.
  • Yo-Pro-1 is a carbocyanine nucleic acid stain that competitively binds to DNA against the polymer.
  • FIG. 10 Gel permeation chromatography data (FIG. 10) highlights how the polymers created are bioreducible. They had higher molecular weight initially, but when each bioreducible polymer (5 mg/mL) was run with or without a reducing agent, dithiothreitol (DTT), at 5 mM concentration, their molecular weight dramatically changed to the constituent monomers. The broader peak at earlier time points for each of the polymers disappears with addition of the reducing agent and there is a concomitant increase in small molecular weight monomers at later time points of the run.
  • DTT dithiothreitol
  • FIG. 11 Competitive binding assays (FIG. 11) show how addition of glutathione, a reducing agent, to a representative bioreducible polymer significantly reduces the binding affinity of the polymer to DNA. This property enables the nanoparticles to release their bioactive agent or cargo more efficiently inside cells.
  • the polymer has weak DNA binding affinity, Yo-Pro-1 binds DNA strongly and emits a strong fluorescent signal. However, when the polymer binds DNA with high affinity, it displaces the Yo-Pro-1 dye and prevents this fluorescent signal.
  • the same polymer, BSS-S4-E8, is shown before and after incubation with glutathione at 0 hrs and at 24 hrs.
  • the 24 hr glutathione incubated curve shows that polymer-DNA binding is dramatically reduced compared to both the 0 hr glutathione sample and the 24 hr glutathione-free sample.
  • a polymer would have strong DNA binding in extracellular oxidizing environments, but low DNA binding and efficient DNA release in the cytoplasm of the cell that is a reducing environment.
  • Transfection efficacy of select bioreducible polymers are shown in FIG. 12. All polymers were transfected at 600 ng/well DNA.
  • the best BSS based polymers (designated BSS-S4-E4 and BSS-S4-E1) obtained signals that were 215X and 5OX higher than untreated wells, respectively.
  • end-capping generic base polymer B4-S5 with two reducible end groups made two new effective polymers, which combine hydrolytic degradation with disulfide reduction of the end groups. These polymers have comparable efficacy to FUGENE HD® and LIPOFECTAMINE 2000TM, and leading degradable polymers. The best bioreducible polymer formulations demonstrate significantly reduced transfection in the presence of 5mM glutathione (all controls unaffected);
  • the presently disclosed subject matter demonstrates that these polymers allow for triggered release in the presence of a reducing environment.
  • the presently disclosed polymers have a triggered decreased in polymer molecular weight and reduced nanoparticle binding.
  • these results demonstrate that the bioreducible function could facilitate nanoparticle unpacking in the cytoplasm.
  • the COS-7 transfection data show that small changes to the structure of the base polymer and to the end-groups drastically alter the ability of the polymer to transfect cells.
  • the presently disclosed polymers have comparable transfection efficacy to leading commercially available transfection agents such as LIPOFECTAMINE 2000TM and FUGENE HD®.
  • the effectiveness of B4-S5-E9 and ElO shows that adding bioreducible functionality to a hydrolytically degradable polymer via change in polymer terminal group is a useful method for improving cargo release.
  • Monomers were purchased from commercial vendors including the following: 1,4-butanediol diacrylate (B4) (Alfa Aesar), 5-amino-1-pentanol (S5) (Alfa Aesar), 2- methy Ip entane- 1,5 -diamine (E4), l-(3-aminopropyl)-4-methylpiperazine (E7), l-(3- aminopropyl)pyrrolidine (E8), 4-aminophenyl disulfide (E9), cystamine (ElO), dimethyl sulfoxide (Sigma- Aldrich), FUGENE HD® (Roche), and LIPOFECTAMINE 2000TM (Invitrogen) and were used as received.
  • B4-butanediol diacrylate B4 (Alfa Aesar), 5-amino-1-pentanol (S5) (Alfa Aesar), 2- methy Ip entane- 1,5 -
  • the base polymer with 1.05:1 ratio 3532 mg of 1,4-butanediol diacrylate (17.8 mmol) was added to 1754 mg of 5-amino-1-pentanol (17.0 mmol).
  • the monomers were reacted in the dark by magnetic stirring in glass scintillation vials in dimethyl sulfoxide (DMSO) at 500 mg/mL.
  • DMSO dimethyl sulfoxide
  • the base polymers at 1 : 1.05 and 1.05:1 monomer ratios were synthesized by stirring at 40°C for 48 hours.
  • the base polymers at the other monomer ratios were synthesized by stirring at 90°C for 24 hours.
  • the diacrylate terminated base polymers were endcapped with amine-containing small molecules E4, E7, E8, E9 and ElO. End-capping reactions were performed in 1.5 mL tubes by adding 320 ⁇ L of 0.5 M amine solution in DMSO to 480 ⁇ L of the base polymer dissolved in DMSO. Polymers were stored at -20°C with desiccant until use. Polymers were analyzed by gel permeation chromatography using a Waters Breeze System and 3 Styragel Columns (7.8 x 300 mm) in series: HR 1, HR 3, and HR 4. The eluent was 95% THF/5% DMSO/0.1 M piperidine and ran at 1 mL/min.
  • B. Cell Culture Epithelial cells were cultured at 37 °C and 5% CO 2 in Dulbecco's modified
  • COS-7 and IMR-90 cells were grown following ATCC recommended protocols and reagents.
  • C0S-7s were grown in Dulbecco's Modified Eagle's Medium (DMEM, ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 units/mL of penicillin and streptomycin (Invitrogen).
  • IMR-90s were grown in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 units/mL of penicillin and streptomycin. Cells were subcultured upon confluence and IMR-90s were used prior to passage eight.
  • EMEM Eagle's Minimum Essential Medium
  • ATCC fetal bovine serum
  • GBM Glioblastoma multiforme
  • Eph4 cells were plated at two different cell densities, 150,000 cells/mL and 170,000 cells/mL in 96-well plates in 100 ⁇ L media and allowed to adhere overnight.
  • 100 ⁇ L of diluted polymer solution was mixed vigorously with 100 ⁇ L of DNA solution in a 96-well plate using a multichannel pipette.
  • the particles were allowed to self-assemble for 10 minutes, following which 20 ⁇ L of each formulation was added to the cells containing 100 ⁇ L of complete media per well in quadruplicate.
  • the cells were incubated with the particles for four hours and then the particles were aspirated with a 12-channel aspirator wand.
  • 110 ⁇ L of diluted polymer solution was mixed vigorously with 110 ⁇ L of DNA solution in an eppendorf tube.
  • the cells were trypsinized for approximately 5 to 10 minutes at 37°C. 500 ⁇ L of complete media with serum was added to each well to neutralize the trypsin. The cell suspension was transferred to a 1.5 mL eppendorf tube and was centrifuged in a microcentrifuge at 1000 rpm for 5 minutes. After aspirating the supernatant, the cell pellet was washed twice with PBS and eventually resuspended in 500 ⁇ L of buffer containing PBS, 1 :20 fetal bovine serum and 1 :200 parts propidium iodide (PI).
  • PI propidium iodide
  • the tube containing the final cell suspension was kept on ice prior to flow cytometry analysis using the FACScan flow cytometry scanner (Flow Cytometry Facility, Johns Hopkins School of Medicine).
  • the flow cytometry results were analysed using the FlowJo software (Tree Star, Inc.).
  • the untreated control sample (with no GFP, no PI) was analyzed to set the gating for the intact cell population in the forward vs. side scatter dot plot and the propidium iodide only control (no GFP) was used to gate the live cell and dead cell population.
  • the results were quantified as % GFP positive cells/live cells.
  • Mammary epithelial cultures were prepared as previously described (A. J. Ewald, et al., "Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis,” Dev Cell. vol. 14(4) pp. 570-581, April 2008).
  • glands were minced and tissue was shaken for 30 min at 37°C in a 50 mL collagenase/trypsin solution in DMEM/F12 (GIBCO-BRL), 0.1 g trypsin (GIBCO- BRL), 0.1 g collagenase (Sigma C5138), 5 mL fetal calf serum, 25 ⁇ L of 10 ⁇ g/mL insulin, and 50 ⁇ L of 50 ⁇ g/mL gentamicin.
  • the collagenase solution was centrifuged at 1500 rpm for 10 min, dispersed through 10 mL DMEM/F12, centrifuged at 1500 rpm for 10 min, and then resuspended in 4 mL DMEM/F12 + 40 ⁇ L DNase (2U/ ⁇ L) (Sigma). The DNase solution was shaken by hand for 2-5 min, then centrifuged at 1500 rpm for 10 min. Organoids were separated from single cells through four differential centrifugations (pulse to 1500 rpm in 10 mL DMEM/F12).
  • FIG. 3 demonstrates that genes encoding green fluorescent protein are delivered to primary cells in three-dimensional organoids by the presently disclosed polymer designated B4-S5-E9 (also referred to herein as Poly 3).
  • FIG. 4 shows that the disulfide end-group, designated herein as E9, can make biphasic polymer designated B4-S5-E9 more effective than other polymers such as those designated as B4-S5-E7 or B4-S5-E8, which have difference end groups designated E7 and E8 herein, at transfecting primary cells in three-dimensional organoids.
  • gene delivery of the presently disclosed polymers designated B4-S5-E10 is also high to as compared to commercially available reagent FUGENE HD® (shown in FIG. 6, % GFP positive as determined by FACS). With the same molecular structure, gene delivery efficacy can also depend strongly on the synthesis conditions used (monomer ratio and temperature).
  • FIG. 5 shows gene delivery with a series of presently disclosed polymers comparable to FUGENE HD® in (top) Hl 46 and (bottom) H446 lung cancer cells.
  • FIG. 7 demonstrates the ability of a presently disclosed polymer designated B4-S5-E10 (60 w/w) for transfecting Glioblastoma Multiforme (GBM) cells as measured by flow cytometry and Green Fluorescent Protein (GFP) expression.
  • FIG. 8 shows this same polymeric nanoparticle formulation with GBM cells by microscopy. Gene delivery and cell viability of polymer B4-S4-E7 with IMR90 cells is shown in FIG. 21.
  • FIG. 22 shows transfection of a retinal neuron with a representative polymer B4-S4-E8. GFP is expressed brightly and morphological structures are good.
  • FIG. 23 shows transfection of a luciferase gene across many representative polymers in COS-7 cells. Each polymer is able to form nanoparticles that deliver genes to COS-7 cells. The polymers were synthesized at 90 °C unless indicated as 40 °C.
  • FIG. 24 shows transfection of a luciferase gene across many representative polymers in IMR-90 cells. Each polymer is able to form nanoparticles that deliver genes to primary human fibroblasts, IMR-90s. The polymers were synthesized at 90 °C unless indicated as 40 °C. Tuning the polymer backbone monomer, side group monomer, terminal group monomer, monomer ratio during synthesis, synthesis temperature, and nanoparticle formulation ratio (w/w) each independently varies overall gene delivery efficacy.
  • these multicomponent polymers described can be designed to either show cell-type specificity towards specific cells or be highly effective for the transfer of nucleic acids intracellularly across a wide range of cells.
  • This example demonstrates the many uses of these materials in vitro as reagents and promisingly suggests their therapeutic utility when used in vivo as one skilled in the art could do through either intravenous, intradermal, subcutaneous, intramuscular, and/or intraperitoneal injection, implantation, or other means.
  • this example also shows that properties of the polymers can be tuned to increase delivery efficacy.
  • changes to multicomponent gene delivery can improve efficacy the following ways: improving protection/encapsulation of a cargo, improving cellular uptake and cell- specific uptake, control of endosomal buffering and endosomal escape, independent control of DNA release, triggered release of an active agent, modification of particle surface charge, increased diffusion through the cytoplasm, active transport through the cytoplasm, increased nuclear import within the cell, and increased transcription and translation of delivered nucleic acids.
  • Changes to cell-specific delivery have been described in this example above and changes to DNA release and triggered release have been described herein in Example 2.
  • the molecular weight can be tuned through three different mechanisms: changing the monomer ratio, changing the reaction temperature and time, adding a cross-linking agent.
  • a range of molecular weights formed with base polymer B4-S5 is shown in FIG. 9 as an example. How these molecular weights can be further tuned by selection of terminal group and/or cross-linker is shown in FIG. 17. Varying these conditions as well as formulation conditions (polymer to DNA mass ratio, mixing rate, buffer composition, and incubation time) can tune nanoparticle size as well. Nanoparticle size is shown in FIG.
  • Nanoparticles formed by self- assembly of B4-S5 -based polymers including B4-S5-E9 and B4-S5-E10 with enhanced green fluorescent protein (EGFP) DNA were sized using two techniques: Dynamic Light Scattering (intensity-weighted mean) and Nanoparticle Tracking Analysis (NTA). NTA was used to determine both the direct number- weighted mean and the mode. Nanoparticles shown in this example are nanoparticles in the useful range for intracellular delivery sized from approximately 50 nm to 250 nm. By tuning the formulation conditions (polymer to DNA mass ratio, mixing rate, buffer composition, and incubation time), these particles also can be made smaller or larger in size up to micron sized particles.
  • the size of the particles can tune the transport of the particles though the cell. Alteration of the polymer terminal group can also change the nanoparticle' s charge, affecting uptake and transport through the cell. Through changes to the multi component structure of the polymers herein, the compartment of delivery, endosomal buffering, and escape from the endosome to the cytoplasm can also be tuned.
  • FIG. 13 shows the titration of representative presently disclosed polymers (e.g., B4-S5-E9, B4-S5-E10, BSS-S5-E7, etc.) showing that they can buffer the pH of the endosomal compartment (pH ⁇ 6) as is needed to protect drug delivery agents and facilitate endosomal rupture through the proton sponge effect.
  • Monomers were purchased from commercial vendors. A two-step polymer synthesis scheme was used. Acrylate-terminated polymer was first synthesized at different acrylate monomer to amine monomer molar ratios, including 1 :1.1, 1 : 1.05, 1 :1, 1.05:1,1.1 :1 and 1.2:1. The monomers were reacted in the dark by magnetic stirring in glass scintillation vials in dimethyl sulfoxide (DMSO) at 500 mg/mL. The base polymers at 1 :1.05 and 1.05:1 monomer ratios were synthesized by stirring at 40°C for 48 hours. The base polymers at the other monomer ratios were synthesized by stirring at 90°C for 24 hours.
  • DMSO dimethyl sulfoxide
  • the diacrylate terminated base polymers were end capped with amine-containing small molecules El and E8. End- capping reactions were performed in 1.5 mL tubes by adding 320 ⁇ L of 0.5 M amine solution in DMSO to 480 ⁇ L of the base polymer dissolved in DMSO. Polymers were stored at -20°C with desiccant until use. Polymers were analyzed by gel permeation chromatography using a Waters Breeze System and 3 Styragel Columns (7.8 x 300 mm) in series: HR 1, HR 3, and HR 4. The eluent was 95% THF/5% DMSO/0.1 M piperidine and ran at 1 mL/min.
  • Particle size was measured by dynamic light scattering on a Malvern Zetasizer Nano ZS (volume averaged size) or by Nanoparticle Tracking Analysis on a Nanosight LMlO (number averaged size).
  • hydrophilic polymer is chosen as the multicomponent material. If a hydrophobic peptide/protein is to be encapsulated than a hydrophobic polymer is chosen. Backbone, side chain, and terminal group can be modified to increase the hydrophobic or hydrophilic character of the polymer as has been described.
  • the peptide/protein to be encapsulated is first dissolved in a suitable solvent such as DMSO or PBS. Then, it is combined with the polymer in sodium acetate (NaAc). This solution is then diluted with either: sodium acetate, OptiMem, DMEM, PBS, or water depending on particle size desired. The solution in vortexed to mix and then left to incubate for 5-15 min for particle assembly to take place. A third component that is amphipathic or that is multivalent (like DNA) was sometimes added to facilitate particle formation and to form tertiary particles.
  • FIG. 20 shows particle sizing data of nanoparticles formed by polymer BL1-S4-E1 and a representative hydrophobic peptide (SPWSPCSTSCGLGVSTRI). Peak of number distribution of nanoparticles is 74 nm for a polymer to peptide mass ratio of 1 : 1. Size is by number as measured by Nanoparticle Tracking Analysis.
  • FIG. 25 shows polymer/peptide particle size depends on polymer structure, formulation conditions including buffer, and peptide that is being encapsulated. All sizing was conducted by dynamic light scattering and volume-averaged sizes are reported. All formulations were at 10 weight polymer to 1 weight peptide.
  • C refers to peptide NGRKACLNPASPIVKKIIEKMLNS and "P” refers to peptide LRRFSTMPFMFCNINNVCNF.
  • P refers to peptide LRRFSTMPFMFCNINNVCNF.
  • nanoparticles formed through these procedures that encapsulate active agents can themselves be encapsulated into a larger microparticle or device.
  • This larger structure can be degradable and can also be not degradable and instead serves as a reservoir that can be refilled with the nanoparticles. Any biomaterial can be used for this larger structure.
  • one method of encapsulation is the double emulsion technique described as following: Aqueous Phase (Prepare sterile): 250 ⁇ L PBS, 2.25 mg BSA, 250 ⁇ g peptide nanoparticles; Organic Phase: 5 mL Dichloromethane, 200 mg PLGA, Homogenizer Phase: 1% PVA in 50 mL water; Final PVA Solution: 0.5% PVA in 100 mL water.
  • Aqueous Phase Prepare sterile
  • BSA 250 ⁇ g peptide nanoparticles
  • Organic Phase 5 mL Dichloromethane, 200 mg PLGA
  • Homogenizer Phase 1% PVA in 50 mL water
  • Final PVA Solution 0.5% PVA in 100 mL water.
  • the first step is forming a primary emulsion with a sonicator (the nanoparticles are in the aqueous phase of this emulsion)
  • the second step is immediately forming a secondary emulsion with a homogenizer
  • the third step is adding the homogenized solution to a 0.5% PVA solution under magnetic stirring.
  • the solvent is left to evaporate in a chemical hood and then the particles are collected following cycles of centrifuging and washing with water.
  • the final particles are lyophilized for 2 days and stored as a powder at -20 C with desiccant. As the microparticles degrade, the nanoparticles will be released allowing sustained release of the nanoparticles.
  • the peptide or protein can be directly coated onto the particle rather than encapsulated within it or may be encapsulated by cross-linked polymers to form a gel or scaffold.
  • the nanoparticle and/or microparticles formed can be further modified by coating with the polyelectrolyte polymers described herein.
  • Monomers were purchased from commercial vendors. A two-step polymer synthesis scheme was used. Acrylate-terminated polymer was first synthesized at different acrylate monomer to amine monomer molar ratios, including 1 :1.1, 1 : 1.05, 1 :1, 1.05:1,1.1 :1 and 1.2:1. The monomers were reacted in the dark by magnetic stirring in glass scintillation vials in dimethyl sulfoxide (DMSO) at 500 mg/mL. The base polymers at 1 :1.05 and 1.05:1 monomer ratios were synthesized by stirring at 40°C for 48 hours. The base polymers at the other monomer ratios were synthesized by stirring at 90°C for 24 hours.
  • DMSO dimethyl sulfoxide
  • the diacrylate terminated base polymers were end capped with amine-containing small molecules El and E8. End- capping reactions were performed in 1.5 mL tubes by adding 320 ⁇ L of 0.5 M amine solution in DMSO to 480 ⁇ L of the base polymer dissolved in DMSO. Polymers were stored at -20°C with desiccant until use. Polymers were analyzed by gel permeation chromatography using a Waters Breeze System and 3 Styragel Columns (7.8 x 300 mm) in series: HR 1, HR 3, and HR 4. The eluent was 95% THF/5% DMSO/0.1 M piperidine and ran at 1 mL/min. Particle size was measured by dynamic light scattering on a Malvern Zetasizer Nano ZS (volume averaged size) or by Nanoparticle Tracking Analysis on a Nanosight LMl 0 (number averaged size). B. Representative Embodiments
  • FIG. 14 shows polymer/siRNA particle size (nm) as a function of formulation conditions (weight ratio polymer to RNA) for a representative presently disclosed polymer designated B5-S3-E9 (1.2: 1 ratio for B5-S3).
  • the formulations of the particles can be tuned to vary biophysical properties of the particle and their release.
  • FIG. 15 shows the particle size/biophysical characterization of polymer/siRNA nanoparticles formed by a presently disclosed polymer designated BL2-S5-E10, 1.2:1.
  • Mean particle size 20 w/w polymer/siRNA 100 nm.
  • FIG. 16 shows direct delivery of siRNA to brain cancer cells using polymeric nanoparticles comprising a presently disclosed polymer designated B5-S3-E9. Brain cancer cells containing FITC-labeled siRNA molecules are shown as bright regions on this image.
  • FIG. 16 shows direct delivery of siRNA to brain cancer cells using polymeric nanoparticles comprising a presently disclosed polymer designated B5-S3-E9. Brain cancer cells containing FITC-labeled siRNA molecules are shown as bright regions on this image.
  • FIG. 7 demonstrates the ability of a presently disclosed polymer designated B4-S5-E10 (60 w/w) for transfecting Glioblastoma Multiforme (GBM) cells as measured by flow cytometry and Green Fluorescent Protein (GFP) expression and FIG. 8 shows a presently disclosed polymer (B4-S5-E10 (60 w/w)) and GBM cells by microscopy.
  • Direct delivery of DNA/indirect delivery of RNA shows that many of these cells actively transcribe and translate this DNA to generate the GFP expression.
  • the delivered plasmid could encode various shRNA molecules that are active within the cell instead of or in addition to GFP. This is an indirect method of RNA delivery in addition to the direct method as demonstrated with siRNA.
  • important design considerations are: different polymer structures, different formulation condition including a lower polymer to nucleic acid weight ratio, and in some cases the addition of a third component that has multivalent characteristics.
  • the particles described above can be combined with nanoparticle targeting (through biomaterial selection, nanoparticle biophysical properties, and/or a targeting ligand) and transcriptional targeting.
  • Transcriptional targeting includes designing a promoter so that the delivered nanoparticles carrying a nucleic acid cargo are only active in the cells or tissue types of interest.
  • Combinations of different genetic cargos and/or particles are co-delivered simultaneously to deliver nucleic acids that both 1) induce apoptosis (genes for tumor necrosis factor-related apoptosis-inducing ligand(TRAIL), p53, etc) and 2) cause differentiation of cancer stem cells (Bone morphogenetic protein 4 (BMP -4) DNA, Glycogen synthase kinase 3beta shRNA/siRNA, etc.).
  • BMP -4 cancer stem cells
  • These nucleic acids are driven by brain cancer specific promoters such as Nestin and Sox-2 for brain cancer stem cells and Glial fibrillary acid protein (GFAP) for glia.

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Abstract

L'invention porte sur des polymères dégradables qui ont été synthétisés et qui s'auto-assemblent avec de l'ADN pour former des particules qui sont efficaces pour l'administration de gène. De petits changements aux conditions de synthèse du polymère, aux conditions de formulation des particules et à la structure du polymère permettent des changements significatifs de l'efficacité d'une manière dépendante du type cellulaire. Les polymères présentés présentement sont plus efficaces que les matières disponibles dans le commerce, telles que la LIPOFECTAMINE 2000™, le FUGENE® ou la polyéthylènimine (PEI) pour l'administration de gène à des fibroblastes cancéreux ou des fibroblastes primaires humains. Les matières divulguées présentement peuvent être utiles pour le traitement thérapeutique d'un cancer et la médecine régénérative.
PCT/US2010/035127 2009-05-15 2010-05-17 Polymères cationiques dégradables multicomposants WO2010132879A2 (fr)

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US13/272,042 US9717694B2 (en) 2009-05-15 2011-10-12 Peptide/particle delivery systems
US14/644,397 US9884118B2 (en) 2009-05-15 2015-03-11 Multicomponent degradable cationic polymers
US15/645,337 US10786463B2 (en) 2009-05-15 2017-07-10 Peptide/particle delivery systems
US15/821,368 US20180177881A1 (en) 2009-05-15 2017-11-22 Multicomponent Degradable Cationic Polymers
US16/895,596 US20200297851A1 (en) 2009-05-15 2020-06-08 Multicomponent degradable cationic polymers
US17/149,583 US20210220287A1 (en) 2009-05-15 2021-01-14 Peptide/particle delivery systems
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US20120114759A1 (en) * 2009-05-15 2012-05-10 The Johns Hopkins University Peptide/particle delivery systems
US10786463B2 (en) 2009-05-15 2020-09-29 The Johns Hopkins University Peptide/particle delivery systems
US20210220287A1 (en) * 2009-05-15 2021-07-22 The Johns Hopkins University Peptide/particle delivery systems
WO2012075217A1 (fr) * 2010-12-02 2012-06-07 The Board Of Regents Of The University Of Idaho Procédé de stimulation de l'ostéogenèse
US8728464B2 (en) 2010-12-02 2014-05-20 University Of Idaho Method for stimulating osteogenesis
WO2014066898A1 (fr) * 2012-10-26 2014-05-01 The Johns Hopkins University Approche couche par couche pour co-administrer l'adn et le petit arn interférent au moyen de nanoparticules d'or (aunps) : une plate-forme potentielle de modification de la cinétique de libération
US11547729B2 (en) 2014-08-07 2023-01-10 The Johns Hopkins University Nanoparticle modification of human adipose-derived mesenchymal stem cells for treating brain cancer and other neurological diseases
US11401380B2 (en) 2015-03-26 2022-08-02 The Johns Hopkins University Poly(β-amino ester)-co-polyethylene glycol (PEG-PBAE-PEG) polymers for gene and drug delivery
US10695427B2 (en) 2015-04-06 2020-06-30 The Johns Hopkins University Shape memory particles for biomedical uses
CN108883150A (zh) * 2015-11-19 2018-11-23 阿斯克雷佩西治疗公司 具有抗血管生成、抗淋巴管生成以及消水肿性质的肽和纳米粒子制剂
US11674959B2 (en) 2017-08-03 2023-06-13 The Johns Hopkins University Methods for identifying and preparing pharmaceutical agents for activating Tie1 and/or Tie2 receptors
US11883541B2 (en) 2017-10-02 2024-01-30 The Johns Hopkins University Nonviral gene transfer to the suprachoroidal space
WO2022159855A1 (fr) * 2021-01-25 2022-07-28 The Johns Hopkins University Nanoparticules polymères bioréductibles photoréticulées pour administration ameliorée d'arn
WO2023131648A1 (fr) 2022-01-05 2023-07-13 Branca Bunus Limited Compositions de nanoparticules pour thérapie génique

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WO2010132879A3 (fr) 2011-03-31
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US20180177881A1 (en) 2018-06-28

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